Surface Coating Structure and Its Interaction with Cytochrome c in EG6-Coated Nanoparticles Varies with Surface Curvature.

The composition, orientation, and conformation of proteins in biomolecular coronas acquired by nanoparticles in biological media contribute to how they are identified by a cell. While numerous studies have investigated protein composition in biomolecular coronas, relatively little detail is known about how the nanoparticle surface influences the orientation and conformation of the proteins associated with them. We previously showed that the peripheral membrane protein cytochrome c adopts preferred poses relative to negatively charged 3-mercaptopropionic acid (MPA)-gold nanoparticles (AuNPs). Here, we employ molecular dynamics simulations and complementary experiments to establish that cytochrome c also assumes preferred poses upon association with nanoparticles functionalized with an uncharged ligand, specifically ω-(1-mercaptounde-11-cyl)hexa(ethylene glycol) (EG6). We find that the display of the EG6 ligands is sensitive to the curvature of the surface-and, consequently, the effective diameter of the nearly spherical nanoparticle core-which in turn affects the preferred poses of cytochrome c.

Lipopolysaccharide Density and Structure Govern the Extent and Distance of Nanoparticle Interaction with Actual and Model Bacterial Outer Membranes.

Design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations and assessment of the potential implications of nanoparticle release into the environment requires understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate the electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the outer leaflet-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed the electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) and second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. The association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides.

Variation of protein corona composition of gold nanoparticles following plasmonic heating.

It is well recognized that the primary interaction of most biological environments with nanoparticles (NPs) is strongly influenced by a long-lived ("hard") protein corona that surrounds the NP and remains strongly adsorbed to its surface. The amount and composition of associated proteins in the corona adsorbed onto the NPs is related to several important factors, including the physicochemical properties of the NPs and the composition of the protein solution. Here, for the first time, it is shown that plasmonic heat induction (by laser activation) leads to significant changes in the composition of the hard protein corona adsorbed on low aspect ratio gold nanorods. Using mass spectrometry, several proteins in the corona were identified whose concentrations change most substantially as a result of photoinduced (plasmonic) heating versus simple thermal heating. Molecular modeling suggests that the origin of these changes in protein adsorption may be the result of protein conformational changes in response to much higher local temperatures that occur near the gold nanorods during photoinduced, plasmonic heating. These results may define new applications in vivo for NPs with hyperthermia capability and better define the likely interactions of cells with NPs after plasmonic heating. Potential changes in the protein corona following hyperthermia treatment may influence the final biological fate of plasmonic NPs in clinical applications and help elucidate safety considerations for hyperthermia applications.

Tuning cellular response to nanoparticles via surface chemistry and aggregation.

The aggregation of gold nanoparticles (Au NPs) in cell media is a common phenomenon that can influence NP-cell interactions. Here, we control Au NP aggregation in cell media and study the impact of Au NP aggregation on human dermal fibroblast (HDF) cells. By first adding Au NPs to fetal bovine serum (FBS) and then subsequently to a buffer, aggregation can be avoided. Aggregation of Au NPs also can be avoided by coating Au NPs with other biomolecules such as lipids. The aggregation state of the Au NPs influences cellular toxicity and Au NP uptake: non-aggregated cationic Au NPs are four-fold less toxic to HDF cells than aggregated cationic Au NPs, and the uptake of non-aggregated anionic citrate Au NPs is three orders of magnitude less than that of aggregated citrate Au NPs. Upon uptake of Au NPs, cellular F-actin fiber formation is disrupted and actin dots are predominant. When lipid-coated Au NPs are doped with a fluorescent lipid (F-lipid) and incubated with HDF cells, the fluorescence from the F-lipid was found throughout the cell, showing that lipids can dissociate from the Au NP surface upon entering the cell.

Homing peptide-conjugated gold nanorods: the effect of amino acid sequence display on nanorod uptake and cellular proliferation.

Gold nanorods (GNRs) have attracted significant interest in the field of medicine as theranostic agents for both imaging and photothermal ablation of cancerous cells/tissues. Targeting theranostic GNRs specifically to cancer cells is necessary to enhance treatment efficacy and minimize undesired side effects. In this study, targeting functionalized GNR to EphA2 receptors that are overexpressed on prostate cancer cells was investigated as a strategy to achieve enhanced GNR uptake by cancer cells. In addition, the influence of targeting peptide orientation on functionalized GNR uptake by PC-3 cells was explored. GNRs of aspect ratio 4 were functionalized with an EphA2 homing peptide, YSA, using a layer-by-layer polypeptide wrapping approach. In parallel, an analogous population of YSA-modified GNRs, which display a reversed YSA peptide, with the N- and C- termini reversed, was also prepared. GC-MS analysis of the YSA-GNRs indicated that functionalized GNRs displayed approximately 3000 peptides/GNR. The functionalized GNRs remained well-dispersed in biological media for short times (<24 h). An increase in GNRs uptake of the YSA-GNRs by PC-3 cells, compared to the reversed YSA-GNRs, was observed under identical incubation conditions. Lastly, the effect of the YSA-GNRs binding to EphA2 receptors on prostate cancer cell proliferation was also studied. The YSA-functionalized GNRs inhibit PC-3 proliferation at a significantly lower effective dose than free YSA. Overall, the polypeptide LBL deposition technique provides a facile route to target nanoparticles to overexpressed cellular receptors, with the caveat that the specific orientation and display of the targeting moiety plays a critical role in the interaction between the nanoparticle and the cell.

The gold standard: gold nanoparticle libraries to understand the nano-bio interface.

Since the late 1980s, researchers have prepared inorganic nanoparticles of many types--including elemental metals, metal oxides, metal sulfides, metal selenides, and metal tellurides--with excellent control over size and shape. Originally many researchers were primarily interested in exploring the quantum size effects predicted for such materials. Applications of inorganic nanomaterials initially centered on physics, optics, and engineering but have expanded to include biology. Many current nanomaterials can serve as biochemical sensors, contrast agents in cellular or tissue imaging, drug delivery vehicles, or even as therapeutics. In this Account we emphasize that the understanding of how nanomaterials will function in a biological system relies on the knowledge of the interface between biological systems and nanomaterials, the nano-bio interface. Gold nanoparticles can serve as excellent standards to understand more general features of the nano-bio interface because of its many advantages over other inorganic materials. The bulk material is chemically inert, and well-established synthetic methods allow researchers to control its size, shape, and surface chemistry. Gold's background concentration in biological systems is low, which makes it relatively easy to measure it at the part-per-billion level or lower in water. In addition, the large electron density of gold enables relatively simple electron microscopic experiments to localize it within thin sections of cells or tissue. Finally, gold's brilliant optical properties at the nanoscale are tunable with size, shape, and aggregation state and enable many of the promising chemical sensing, imaging, and therapeutic applications. Basic experiments with gold nanoparticles and cells include measuring the toxicity of the particles to cells in in vitro experiments. The species other than gold in the nanoparticle solution can be responsible for the apparent toxicity at a particular dose. Once the identity of the toxic agent in nanoparticle solutions is known, researchers can employ strategies to mitigate toxicity. For example, the surfactant used at high concentration in the synthesis (0.1 M) of gold nanorods remains on their surface in the form of a bilayer and can be toxic to certain cells at 200 nM concentrations. Several strategies can alleviate the toxic response. Polyelectrolyte layer-by-layer wrapping can cover up the surfactant bilayer, or researchers can exchange the surfactant with chemically similar molecules. Researchers can also replace the surfactant with a biocompatible thiol or use a polymerizable surfactant that can be "stitched" onto the nanorods and reduce its lability. In all these cases, however, proteins or other molecules from the cellular media cover the engineered surface of the nanoparticles, which can drastically change the charges and functional groups on the nanoparticle surface.

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy.

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.

Nanoparticle-protein interactions: a thermodynamic and kinetic study of the adsorption of bovine serum albumin to gold nanoparticle surfaces.

Investigating the adsorption process of proteins on nanoparticle surfaces is essential to understand how to control the biological interactions of functionalized nanoparticles. In this work, a library of spherical and rod-shaped gold nanoparticles (GNPs) was used to evaluate the process of protein adsorption to their surfaces. The binding of a model protein (bovine serum albumin, BSA) to GNPs as a function of particle shape, size, and surface charge was investigated. Two independent comparative analytical methods were used to evaluate the adsorption process: steady-state fluorescence quenching titration and affinity capillary electrophoresis (ACE). Although under favorable electrostatic conditions kinetic analysis showed a faster adsorption of BSA to the surface of cationic GNPs, equilibrium binding constant determinations indicated that BSA has a comparable binding affinity to all of the GNPs tested, regardless of surface charge. BSA was even found to adsorb strongly to GNPs with a pegylated/neutral surface. However, these fluorescence titrations suffer from significant interference from the strong light absorption of the GNPs. The BSA-GNP equilibrium binding constants, as determined by the ACE method, were 10(5) times lower than values determined using spectroscopic titrations. While both analytical methods could be suitable to determine the binding constants for protein adsorption to NP surfaces, both methods have limitations that complicate the determination of protein-GNP binding constants. The optical properties of GNPs interfere with Ka determinations by static fluorescence quenching analysis. ACE, in contrast, suffers from material compatibility issues, as positively charged GNPs adhere to the walls of the capillary during analysis. Researchers seeking to determine equilibrium binding constants for protein-GNP interactions should therefore utilize as many orthogonal techniques as possible to study a protein-GNP system.

Applications of colloidal inorganic nanoparticles: from medicine to energy.

The synthesis of well-defined inorganic nanoparticles in colloidal solution, which evolved gradually from the 1950s onward, has now reached the point where applications in both the research world and the wider world can be realized. This Perspective explores some of the successes and still-remaining challenges in nanoparticle synthesis and ligand analysis, highlights selected work in the areas of biomedicine and energy conversion that are enabled by colloidal nanomaterials, and discusses technical barriers that need to be overcome by chemists and other scientists in order for nanotechnology to achieve its promise.

Interactions between natural organic matter and gold nanoparticles stabilized with different organic capping agents.

The adsorption of natural organic matter (NOM) to the surfaces of natural colloids and engineered nanoparticles is known to strongly influence, and in some cases control, their surface properties and aggregation behavior. As a result, the understanding of nanoparticle fate, transport, and toxicity in natural systems must include a fundamental framework for predicting such behavior. Using a suite of gold nanoparticles (AuNPs) with different capping agents, the impact of surface functionality, presence of natural organic matter, and aqueous chemical composition (pH, ionic strength, and background electrolytes) on the surface charge and colloidal stability of each AuNP type was investigated. Capping agents used in this study were as follows: anionic (citrate and tannic acid), neutral (2,2,2-[mercaptoethoxy(ethoxy)]ethanol and polyvinylpyrrolidone), and cationic (mercaptopentyl(trimethylammonium)). Each AuNP type appeared to adsorb Suwannee River Humic Acid (SRHA) as evidenced by measurable decreases in zeta potential in the presence of 5 mg C L(-1) SRHA. It was found that 5 mg C L(-1) SRHA provided a stabilizing effect at low ionic strength and in the presence of only monovalent ions while elevated concentrations of divalent cations lead to enhanced aggregation. The colloidal stability of the NPs in the absence of NOM is a function of capping agent, pH, ionic strength, and electrolyte valence. In the presence of NOM at the conditions examined in this study, the capping agent is a less important determinant of stability, and the adsorption of NOM is a controlling factor.

Direct synthesis of large water-soluble functionalized gold nanoparticles using Bunte salts as ligand precursors.

The widespread use of functionalized gold nanoparticles (AuNPs) in a rapidly increasing number of sensing and biomedical applications has made the development of synthetic methods that combine precise surface chemistry control (functionality) with effective core size control over the range of 1-20 nm crucial. Although a variety of effective methods exist for controlling the core size and functionality during gold nanoparticle synthesis, there is a lack of synthetic methods that permit the direct synthesis of thiol-protected gold nanoparticles with core diameters greater than 5.0 nm. Inspired by previous reports on the use of alkyl thiosulfates (Bunte salts) as ligand precursors, we anticipated that the slow passivation kinetics of these masked thiols would provide a method to synthesize large functionalized AuNPs directly. We found that Bunte salts produce larger AuNPs under the same synthesis conditions than do thiols. We investigated the effect of the ligand/gold ratio, temperature, and reducing agent concentration on the particle diameter and dispersity to understand better how to control particle size. The AuNP core size can be systematically controlled by varying the ratio of ligand precursor/gold (L/Au) and the temperature of the reaction. The synthesis produces functionalized AuNPs ranging from 1.5 to 20.0 nm in diameter. The use of Bunte salts provides a convenient synthetic platform for the synthesis of AuNPs across this size range that possess a variety of surface functionalities, including positive, negative, and neutral functional groups.

Bare surface of gold nanoparticle induces inflammation through unfolding of plasma fibrinogen.

The surface of nanoparticles (NPs) get coated by a wide range of biomolecules, upon exposure to biological fluids. It is now being increasingly accepted that NPs with particular physiochemical properties have a capacity to induce conformational changes to proteins and therefore influence their biological fates, we hypothesized that the gold NP's metal surface may also be involved in the observed Fg unfolding and inflammatory response. To mechanistically test this hypothesis, we probed the interaction of Fg with gold surfaces using molecular dynamic simulation (MD) and revealed that the gold surface has a capacity to induce Fg conformational changes in favor of inflammation response. As the integrity of coatings at the surface of ultra-small gold NPs are not thorough, we also hypothesized that the ultra-small gold NPs have a capacity to induce unfolding of Fg regardless of the composition and surface charge of their coatings. Using different surface coatings at the surface of ultra-small gold NPs, we validated this hypothesis. Our findings suggest that gold NPs may cause unforeseen inflammatory effects, as their surface coatings may be degraded by physiological activity.

Cascading Effects of Nanoparticle Coatings: Surface Functionalization Dictates the Assemblage of Complexed Proteins and Subsequent Interaction with Model Cell Membranes.

Interactions of functionalized nanomaterials with biological membranes are expected to be governed by not only nanoparticle physiochemical properties but also coatings or "coronas" of biomacromolecules acquired after immersion in biological fluids. Here we prepared a library of 4-5 nm gold nanoparticles (AuNPs) coated with either ω-functionalized thiols or polyelectrolyte wrappings to examine the influence of surface functional groups on the assemblage of proteins complexing the nanoparticles and its subsequent impact on attachment to model biological membranes. We find that the initial nanoparticle surface coating has a cascading effect on interactions with model cell membranes by determining the assemblage of complexing proteins, which in turn influences subsequent interaction with model biological membranes. Each type of functionalized AuNP investigated formed complexes with a unique ensemble of serum proteins that depended on the initial surface coating of the nanoparticles. Formation of protein-nanoparticle complexes altered the electrokinetic, hydrodynamic, and plasmonic properties of the AuNPs. Complexation of the nanoparticles with proteins reduced the attachment of cationic AuNPs and promoted attachment of anionic AuNPs to supported lipid bilayers; this trend is observed with both lipid bilayers comprising 100% zwitterionic phospholipids and those incorporating anionic phosphatidylinositol. Complexation with serum proteins led to attachment of otherwise noninteracting oligo(ethylene glycol)-functionalized AuNPs to bilayers containing phosphatidylinositol. These results demonstrate the importance of considering both facets of the nano-bio interface: functional groups displayed on the nanoparticle surface and proteins complexing the nanoparticles influence interaction with biological membranes as does the molecular makeup of the membranes themselves.

Quantitative determination of ligand densities on nanomaterials by X-ray photoelectron spectroscopy.

X-ray photoelectron spectroscopy (XPS) is a nearly universal method for quantitative characterization of both organic and inorganic layers on surfaces. When applied to nanoparticles, the analysis is complicated by the strong curvature of the surface and by the fact that the electron attenuation length can be comparable to the diameter of the nanoparticles, making it necessary to explicitly include the shape of the nanoparticle to achieve quantitative analysis. We describe a combined experimental and computational analysis of XPS data for molecular ligands on gold nanoparticles. The analysis includes scattering in both Au core and organic shells and is valid even for nanoparticles having diameters comparable to the electron attenuation length (EAL). To test this model, we show experimentally how varying particle diameter from 1.3 to 6.3 nm leads to a change in the measured AC/AAu peak area ratio, changing by a factor of 15. By analyzing the data in a simple computational model, we demonstrate that ligand densities can be obtained, and, moreover, that the actual ligand densities for these nanoparticles are a constant value of 3.9 ± 0.2 molecules nm(-2). This model can be easily extended to a wide range of core-shell nanoparticles, providing a simple pathway to extend XPS quantitative analysis to a broader range of nanomaterials.

Effects of charge and surface ligand properties of nanoparticles on oxidative stress and gene expression within the gut of Daphnia magna.

Concern has been raised regarding the current and future release of engineered nanomaterials into aquatic environments from industry and other sources. However, not all nanomaterials may cause an environmental impact and identifying which nanomaterials may be of greatest concern has been difficult. It is thought that the surface groups of a functionalized nanoparticles (NPs) may play a significant role in determining their interactions with aquatic organisms, but the way in which surface properties of NPs impact their toxicity in whole organisms has been minimally explored. A major point of interaction of NPs with aquatic organisms is in the gastrointestinal tract as they ingest particulates from the water column or from the sediment. The main goal of this study was to use model gold NP (AuNPs) to evaluate the potential effects of the different surfaces groups on NPs on the gut of an aquatic model organism, Daphnia magna. In this study, we exposed daphnids to a range of AuNPs concentrations and assessed the impact of AuNP exposure in the daphnid gut by measuring reactive oxygen species (ROS) production and expression of genes associated with oxidative stress and general cellular stress: glutathione S-transferase (gst), catalase (cat), heat shock protein 70 (hsp70), and metallothionein1 (mt1). We found ROS formation and gene expression were impacted by both charge and the specific surface ligand used. We detected some degree of ROS production in all NP exposures, but positively charged AuNPs induced a greater ROS response. Similarly, we observed that, compared to controls, both positively charged AuNPs and only one negatively AuNP impacted expression of genes associated with cellular stress. Finally, ligand-AuNP exposures showed a different toxicity and gene expression profile than the ligand alone, indicating a NP specific effect.

Impacts of gold nanoparticle charge and ligand type on surface binding and toxicity to Gram-negative and Gram-positive bacteria.

Although nanomaterials facilitate significant technological advancement in our society, their potential impacts on the environment are yet to be fully understood. In this study, two environmentally relevant bacteria, Shewanella oneidensis and Bacillus subtilis, have been used as model organisms to elucidate the molecular interactions between these bacterial classes and Au nanoparticles (AuNPs) with well-controlled and well-characterized surface chemistries: anionic 3-mercaptopropionic acid (MPA), cationic 3-mercaptopropylamine (MPNH2), and the cationic polyelectrolyte poly(allylamine hydrochloride) (PAH). The data demonstrate that cationic, especially polyelectrolyte-wrapped AuNPs, were more toxic to both the Gram-negative and Gram-positive bacteria. The levels of toxicity observed were closely related to the percentage of cells with AuNPs associated with the cell surface as measured in situ using flow cytometry. The NP concentration-dependent binding profiles were drastically different for the two bacteria strains, suggesting the critical role of bacterial cell surface chemistry in determining nanoparticle association, and thereby, biological impact.

A simple millifluidic benchtop reactor system for the high-throughput synthesis and functionalization of gold nanoparticles with different sizes and shapes.

Despite the continuing interest in the applications of functionalized nanomaterials, the controlled and reproducible synthesis of many important functionalized nanoparticles (NPs) above the milligram scale continues to be a significant challenge. The synthesis of functionalized NPs in automated reactors provides a viable approach to circumvent some of the shortcomings of traditional nanomaterial batch syntheses, providing superior control over reagent addition, improved reproducibility, the opportunity to interface real-time product monitoring, and a viable high-throughput synthetic approach. Here, we demonstrate the construction and operation of a simple millifluidic reactor assembled entirely from commercially available components found in almost any chemical laboratory. This reactor facilitates the aqueous gram-scale synthesis of a variety of functionalized gold nanoparticles, including the synthesis of gold nanospheres with tightly controlled core diameters and gold nanorods with controlled aspect ratios between 1.5 and 4.0. The absolute dimensions (i.e., the transverse diameter) of gold nanorods synthesized within the reactor can also be tailored to produce different gold nanorod shapes, including "small" gold nanorods and gold nanocubes. In addition, we show that the reactor can interface with existing purification and monitoring techniques in order to enable the high-throughput functionalization/purification of gold nanorods and real-time monitoring of gold nanoparticle products for quality control. We anticipate that this millifluidic reactor will provide the blueprint for a versatile and portable approach to the gram-scale synthesis of monodisperse, hydrophilically functionalized metal NPs that can be realized in almost any chemistry research laboratory.